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Recent applications of NGS sequencing in cancer studies. Andrew Gentles CCSB NGS workshop September 2012. You’ve slogged through QC, trimming, alignment, realignment, variant calling. What next ?.
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Recent applications of NGS sequencing in cancer studies Andrew Gentles CCSB NGS workshop September 2012
You’ve slogged through QC, trimming, alignment, realignment, variant calling What next ?
Mutational processes molding the genomes of 21 breast cancers/The life history of 21 breast cancers • Nik-Zainal et al. (2012) Cell 149(5):994-1007 • Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia • Jan et al. (2012) Sci Trans Med 4, 149ra118 • Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotatedlincRNA implicated in disease progression • Prensner et al. (2011) Nat Biotech 29: 742-9
Whole genome sequencing of 21 Breast cancers >30x coverage tumor and normal (188x for *)
Analysis outline • WGS sequencing to >30x coverage tumor/normal • ~100 bp paired-end reads • BWA alignment • Compare tumor/normal for variant calling • CaVEMan, Pindel • Detection of structural rearrangements • In-house method • Inference of copy number changes • ASCAT
Summary of somatic mutations • 183916 somatic mutations (SNVs) identified in total • 1372 missense, 117 nonsense, 2 stop-lost, 37 splice, 521 silent • Most frequent mutations in known cancer genes such as TP53, GATA3, PIK3CA, MAP2K4, SMAD4, MLL2, MLL3, NCOR1
Higher rate in BRCA1/2 C>A most common Mutational spectrum in breast cancer
A very deep look into mutation frequencies to reconstruct tumor evolution
PD4120a • 188x coverage – enables deep look at mutation frequencies • 70690 somatic substitutions • Some in <5% of reads • Mainly C>* in TpC context • High rate of validation
Patterns of copy number alteration in PD4120a Relatively few CNVs Some sub-clonal
Mutation frequencies show clusters representing major and minor clones 2 1 D 2 3 C 2 B A 35% of reads -> all tumor cells since tumor is 70% tumor (cluster D) Trisomy 1q early since few mutations with high read fraction – most are subclonal 3 major clusters of sub-clonal mutations (A,B,C)
15600 26762 5% 11% 19% 35% Founder clone “most-recent common ancestor”
D 4 C B A Cluster C ~19% - more than half of tumor cells (since >1/2*35%) “Pigeonhole principle”: for any 2 mutations, at least one tumor cell must have both – must be on same part of phylogenetic tree If one such mutation in greater fraction than another, must have occurred earlier Cluster C must be on same phylogenetic branch as del13
If SNVs close enough to SNPs, can be phased with them • 2171 on chr13 • 756 can be phased
Phasing of somatic mutations (Supp Fig 4) Found 17 mutually exclusive, 76 examples of sub-clonal evolution
Figure 3: Reconstructed evolution of tumor (see paper for details)
Prospective separation of residual HSC from leukemic patients
Strategy for identifying pre-leukemic mutations in HSC 67-239x exome coverage
Occurrence of AML mutations in residual HSC ~25000x targeted coverage
HSC with the pre-leukemic mutations are capable of differentiating to produce functional immune cells
Enrichment of histone modification marks around transcripts H3K4me2 H3K4me3 Figure 2
PCAT-1 is highly expressed in metastatic/high-grade prostate cancer Figure 4b Figure 3f PCAT-1 expression is mutually exclusive with EZH2
RNA-seq discovers novel ncRNAs • PCAT-1 highly expressed in high grade/metastatic prostate cancer • PCAT-1 promotes proliferation • Hypothesized role with EZH2 (c.f. HOTAIR)
Final items • Please fill out evaluation form! • Slides: • Available soon from http://ccsb.stanford.edu • Sequence answers forum: • http://seqanswers.com • Stanford discussion group • https://mailman.stanford.edu/mailman/listinfo/wgs_club_stanford
